Traditionally, the sol-gel method has been used to synthesize organic-inorganic composites including enzymes to improve the stability of the enzymes in organic or aqueous solutions. In most applications, silica-encapsulated enzymes are synthesized in two stages by the sol-gel method. In the first stage, silica monomers are hydrolyzed in an aqueous solution containing enzymes. In the second stage, the hydrolyzed silica monomers encapsulate the enzymes as they are condensed, thus forming a growing silica network. Through this procedure, a sol-gel matrix in which silica is hydrolyzed and condensed is formed. However, the sol-gel matrix approach for encapsulating enzymes induces mass transfer limitation against substrates diffusion through the thick sol-gel matrix.
Enzyme encapsulation is frequently used as one of the enzyme immobilization methods for caging and stabilization of the enzymes. However, according to most researches, sol-gel composites in a few micrometers to millimeters range restricted the transfer of substrate molecules from medium to active sites of enzymes. Accordingly, the sol-gel encapsulation method could stabilize the individual enzyme molecules while increasing a Michaelis-Menten constant, so draws a lot of attentions. Another general approach is to form reverse micelles. However, the reverse micelles are including large quantity of water molecules, which can form sol-gel matrix via silica hydrolysis and condensation. Consequently, the thick sol-gel matrix causes serious mass transfer limitation against substrates diffusion from medium to active site of enzyme.
Currently, it is reported that single enzyme nanoparticles (SENs) are obtained by forming an organic-inorganic hybrid polymer network of less than a few nanometers thick without entirely encapsulating the enzyme aggregates. Specifically, the synthesis of the SENs begins with solubilization of enzyme molecules in a hexane solution. The solubilization process, which uses smaller amount of surfactant than the reverse micelle approach, involves extracting individual enzyme molecules with the organic solvent while preventing aggregation of the enzymes. The surface of the solubilized enzymes is exposed to the organic solvent together with the minimal amount of water molecules. This is very important for successfully forming SENs with a very thin network. However, the synthesis of the SENs necessarily requires formation of the functional groups on the surface of the enzymes in hexane and polymerizing them by using the functional groups, for example, vinyl groups. Since the radical polymerization process is very complicated and sensitive to reaction condition, it is very difficult to produce SENs in a large scale.